Purification of hbv antigens for use in vaccines

ABSTRACT

The present invention relates to a method for the production of a hepatitis B antigen suitable for use in a vaccine, the method comprising purification of the antigen in the presence of cysteine, to vaccines comprising such antigens.

[0001] This invention relates to a novel process of manufacture of ahepatitis B vaccine for use in the treatment or prophylaxis of hepatitisB virus (HBV) infections. It further relates to a HBV vaccine obtainableby the novel process of the invention.

[0002] Chronic hepatitis B virus (HBV) infection, for which there iscurrently limited treatment, constitutes a global public health problemof enormous dimensions. Chronic carriers of HBV, estimated to numbermore than 300 million world-wide, are at risk for development of chronicactive hepatitis, cirrhosis and primary hepatocellular carcinoma.

[0003] Many vaccines which are currently available require apreservative to prevent deterioration. A frequently used preservative isthiomersal which is a mercury-containing compound. Some concerns havebeen raised about the use of mercury in vaccines, although commentatorshave stressed that the potential hazards of thiomersal-containingvaccines should not be overstated (Offit; P. A. JAMA Vol.283;No:16).Nevertheless it would be advantageous to find new and potentially safermethods of preparation of vaccines to replace the use of thiomersal inthe manufacturing process. There is thus a need to develop vaccineswhich are thiomersal-free, in particular hepatitis B vaccines.

[0004] In a first aspect the present invention provides a method forproducing a hepatitis B antigen suitable for use in a vaccine, themethod comprising purification of the antigen in the presence of areducing agent comprising a free —SH group.

[0005] The present invention preferably provides a method of producing astable hepatitis B antigen without trace of thiomersal which comprisespurification of the antigen in the presence of a reducing agent having afree —SH group.

[0006] The antigen preparation is generally without trace of thiomersalwhen thiomersal is not detectable in the purified antigen product usingabsorption spectrophotometery of mercury, as described herein.

[0007] The hepatitis antigen preparation preferably comprises less than0.025 μg mercury per 20 μg protein, suitably as measured by absorptionspectrophotometery.

[0008] Preferably the purification is carried out in the absence ofthiomersal, and the purified antigen is completely free of thiomersal.

[0009] Preferably the antigen is stable, suitably substantially asstable as a hepatitis antigen purified in the presence of thiomersal, asoutlined in Example 1 herein for example.

[0010] Preferably the hepatitis antigen is immunogenic.

[0011] Preferably the reducing agent is added during the antigenpurification process, preferably after growth of cells expressing theantigen.

[0012] Preferably the reducing agent is cysteine, dithiothreitol,β-mercaptoethanol or glutathione, with cysteine being most preferred.

[0013] Accordingly the present invention preferably provides a method ofproducing a stable immunogenic hepatitis B antigen without trace ofthiomersal which comprises purification of the antigen in the presenceof cysteine.

[0014] Preferably the purification is carried out in the presence of acysteine solution.

[0015] Preferably, the cysteine, in solution or powder form, is addedduring the process to a final concentration of between 1 and 10 mM,preferably 1 to 5 mM. More preferably, the cysteine is added to a finalconcentration of about 2 mM.

[0016] Preferably the cysteine is L-cysteine.

[0017] The invention further provides a method of producing a stablehepatitis B antigen without trace of thiomersal wherein the crudeantigen is subjected to gel permeation chomatography, subjected toion-exchange chromatography and mixed with a reducing agent having afree —SH group.

[0018] Preferably the ion-exchange chromatography is anion-exchangechromatography.

[0019] The invention further provides a hepatitis B antigen free ofthiomersal obtainable by the method of manufacture of the presentinvention wherein the antigen is at least as immunogenic and antigenicas the hepatitis B antigen manufactured in the presence of thiomersal.

[0020] The invention further provides an immunogenic hepatitis B antigenhaving a mean ELISA protein ratio greater than 1.5 and an RF1 contentwith at least a 3-fold lower IC50 value than that of the hepatitis Bsurface antigen manufactured in the presence of thiomersal.

[0021] In an further aspect the invention relates to a method for theproduction of a hepatitis antigen suitable for use in a vaccine, themethod comprising purification of the antigen in the presence ofthiomersal and subsequent treatment of antigen in the presence of areducing agent comprising a free —SH group.

[0022] Suitably the treatment is followed by a purification step such asa diaysis step to remove thiomersal.

[0023] Preferably the reducing agent is cysteine, DTT, glutathione or2-mercaptoethanol.

[0024] The hepatitis B antigen of the invention may be used for eitherthe treatment or prophylaxis of hepatitis B infections, especiallytreatment or prophylaxis, for example, of chronic hepatitis Binfections.

[0025] The present invention further provides a vaccine formulationcomprising a hepatitis B antigen of the present invention in conjunctionwith an adjuvant. Preferably the adjuvant is an aluminium salt or apreferential stimulator of TH1 cell response.

[0026] Preferably the antigen is a hepatitis B surface antigen.

[0027] The preparation of hepatitis B surface antigen is welldocumented. See for example, Harford et. al. in Develop. Biol. Standard54, page 125 (1983), Gregg et. al. in Biotechnology, 5, page 479 (1987),EP-A- 0 226 846, EP-A-0 299 108 and references therein.

[0028] As used herein the expression ‘hepatitis B surface antigen’ or‘HBsAg’ includes any HBsAg antigen or fragment thereof displaying theantigenicity of HBV surface antigen. It will be understood that inaddition to the 226 amino acid sequence of the HBsAg S antigen (seeTiollais et. al. Nature, 317, 489 (1985) and references therein) HBsAgas herein described may, if desired, contain all or part of a pre-Ssequence as described in the above references and in EP-A- 0 278 940.HBsAg as herein described can also refer to variants, for example the‘escape mutant’ described in WO 91/14703.

[0029] HBsAg may also refer to polypeptides described in EP 0 198 474 orEP 0 304 578.

[0030] Normally the HBsAg will be in particle form. In a particularlypreferred embodiment the HbsAg will consist essentially of the HbsAgS-antigen mentioned hereinabove.

[0031] The vaccine may advantageously include a pharmaceuticallyacceptable excipient such as a suitable adjuvant. Suitable adjuvants arecommercially available such as, for example, Freund's IncompleteAdjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.);Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2(SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as aluminumhydroxide gel (alum) or aluminum phosphate; salts of calcium, iron orzinc; an insoluble suspension of acylated tyrosine; acylated sugars;cationically or anionically derivatized polysaccharides;polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A andquil A. Cytokines, such as GM-CSF or interleukin-2, -7, or -12, may alsobe used as adjuvants.

[0032] In the formulations of the invention it is preferred that theadjuvant composition induces an immune response predominantly of the TH1type. High levels of Th1-type cytokines (e.g., IFN-γ, TNFα, IL-2 andIL-12) tend to favour the induction of cell mediated immune responses toan administered antigen. Within a preferred embodiment, in which aresponse is predominantly Th1-type, the level of Th1-type cytokines willincrease to a greater extent than the level of Th2-type cytokines. Thelevels of these cytokines may be readily assessed using standard assays.For a review of the families of cytokines, see Mosmann and Coffman, Ann.Rev. Immunol. 7:145-173, 1989.

[0033] Accordingly, suitable adjuvants for use in eliciting apredominantly Th1-type response include, for example a combination ofmonophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipidA (3D-MPL) together with an aluminium salt. Other known adjuvants whichpreferentially induce a TH1 type immune response include CpG containingoligonucleotides. The oligonucleotides are characterised in that the CpGdinucleotide is unmethylated. Such oligonucleotides are well known andare described in, for example WO 96/02555. Immunostimulatory DNAsequences are also described, for example, by Sato et al., Science273:352, 1996. Another preferred adjuvant is a saponin, preferably QS21(Aquila Biopharmaceuticals Inc., Framingham, Mass.), which may be usedalone or in combination with other adjuvants. For example, an enhancedsystem involves the combination of a monophosphoryl lipid A and saponinderivative, such as the combination of QS21 and 3D-MPL as described inWO 94/00153, or a less reactogenic composition where the QS21 isquenched with cholesterol, as described in WO 96133739. Other preferredformulations comprise an oil-in-water emulsion and tocopherol. Aparticularly potent adjuvant formulation involving QS21, 3D-MPL andtocopherol in an oil-in-water emulsion is described in WO 95/17210.

[0034] A particularly potent adjuvant formulation involving QS21, 3D-MPL& tocopherol in an oil in water emulsion is described in WO 95117210 andis a preferred formulation.

[0035] Accordingly in one embodiment of the present invention there isprovided a vaccine comprising a hepatitis B surface antigen of thepresent invention, which additionally comprises a TH-1 inducingadjuvant. A preferred embodiment is a vaccine in which the TH-1 inducingadjuvant is selected from the group of adjuvants comprising: 3D-MPL,QS21, a mixture of QS21 and cholesterol, and a CpG oligonucleotide.Another preferred embodiment is a vaccine comprising a hepatitis Bsurface antigen adjuvanted with a monophosphoryl lipid A or derivativethereof, QS21 and tocopherol in an oil in water emulsion.

[0036] Preferably the vaccine additionally comprises a saponin, morepreferably QS21. Another particular suitable adjuvant formulationincluding CpG and a saponin is described in WO 00/09159 and is apreferred formulation. Most preferably the saponin in that particularformulation is QS21. Preferably the formulation additionally comprisesan oil in water emulsion and tocopherol.

[0037] The present invention further provides a vaccine formulationcomprising a hepatitis B surface antigen of the present invention inconjunction with an adjuvant and additionally comprising one or moreantigens selected from the group consisting of: diptheria toxoid (D),tetanus toxoid (T) acellular pertussis antigens (Pa), inactivated poliovirus (IPV), haemophilus influenzae antigen (Hib), hepatitis A antigen,herpes simplex virus (HSV), chlamydia, GSB, HPV, streptococcuspneumoniae and neisseria antigens. Antigens conferring protection forother diseases may also be combined in the vaccine formulation of thepresent invention.

[0038] In one particular embodiment, the vaccine formulation comprises ahepatitis B surface antigen obtainable by the method of manufacture ofthe present invention in conjunction with an adjuvant and an inactivatedpolio virus.

[0039] The present invention also provides a method of treatment and/orprophylaxis of hepatitis B virus infections, which comprisesadministering to a human or animal subject, suffering from orsusceptible to hepatitis B virus infection, a safe and effective amountof a vaccine of the present invention for the prophylaxis and/ortreatment of hepatitis B infection.

[0040] The invention further provides the use of a hepatitis B surfaceantigen of the present invention in the manufacture of a medicament forthe treatment of patients suffering from a hepatitis B virus infection,such as chronic hepatitis B virus infection.

[0041] The vaccine of the present invention will contain animmunoprotective quantity of the antigen and may be prepared byconventional techniques.

[0042] Vaccine preparation is generally described in PharnaceuticalBiotechnology, Vol.61 Vaccine Design—the subunit and adjuvant approach,edited by Powell and Newman, Plenurn Press, 1995. New Trends andDevelopments in Vaccines, edited by Voller et al., University ParkPress, Baltimore, Md., U.S.A. 1978. Encapsulation within liposomes isdescribed, for example, by Fullerton, U.S. Pat. No. 4,235,877.Conjugation of proteins to macromolecules is disclosed, for example, byLikhite, U.S. Pat. No. 4,372,945 and by Armor et al., U.S. Pat. No.4,474,757. Use of Quil A is disclosed by Dalsgaard et al., Acta VetScand, 18:349 (1977). 3D-MPL is available from Ribi immunochem, USA, andis disclosed in British Patent Application No. 2220211 and U.S. Pat. No.4,912,094. QS21 is disclosed in U.S. Pat. No. 5,057,540.

[0043] The present invention is illutrated by but not limited to thefollowing examples, wherein:

[0044]FIG. 1 illustrates the thiomersal free production process forEngerix B™;

[0045]FIG. 2 illustrates SDS-PAGE analysis of bulk antigen lots; and

[0046]FIG. 3 illustrates residual yeast proteins in bilk antigen lotsproduced by the thiomersal free process.

EXAMPLE 1 Production Process of Hepatitis B Surface Antigen in thePresence of Thiomersal

[0047] The Hepatitis B surface antigen (HBsAg) of SB Biologicalshepatitis B monovalent vaccine (Engerix B™) is expressed as arecombinant protein in Saccharomyces cerevisiae (see Harford et. al.loc. cit.). The 24 kD protein is produced intracellularly andaccumulated in the recombinant yeast cells. At the end of thefermentation the yeast cells are harvested and disrupted in the presenceof a mild surfactant such as Tween 20 to liberate the desired protein.Subsequently the cell homogenate, containing the soluble surface antigenparticles, is prepurified in a series of precipitations and thenconcentrated via ultrafiltration.

[0048] Further purification of the recombinant antigen is performed insubsequent chromatographic separations. In a first step the crudeantigen concentrate is subjected to gel permeation chromatography onSepharose 4B medium. Thiomersal is present in the elution buffer at the4B gel permeation chromatography step. The elution buffer has thefollowing composition: 10 mM Tris, 5% ethylene glycol, pH 7.0, 50 mg/Lthiomersal. Thiomersal is included in this buffer to control bioburden.Most of this thiomersal is removed during the subsequent purificationsteps including ion exchange chromatography, ultracentrifugation anddesalting (gel permeation) so that purified bulk antigen preparationsprepared by the original process contain about 1.2 μg and less than 2 μgof thiomersal per 20 μg of protein.

[0049] An Ion-Exchange chromatography step is performed using aDEAE-matrix and this pool is then subjected to a Cesium-gradientultracentrifugation on 4 pre-established layers of different Cesiumchloride concentrations. The antigen particles are separated fromcontaminating cell constituents according to their density in thegradient and eluted at the end of the centrifugation process. Cesiumchloride is then removed from this pool by a second gel permeation onSepharose gel.

[0050] When HBsAg is prepared by the process containing thiomersal inthe 4B gel permeation buffer, protein concentrations of over 30 mg/mlare recovered in the pooled HBsAg containing fractions from the CsClgradient, corresponding to an equivalent concentration of HBsAg asassayed by the AUSZYME kit from Abbott Laboratories.

[0051] The CsCl ultracentrifugation step usefully eliminates residuallipids, DNA and minor protein contaminants from the HBsAg preparation.It is performed by zonal centrifugation in a Ti 15 rotor from BeckmanInstruments, Fullerton, Calif. at a speed of 30,000 rpm for about 40 to60 hours. The sample to be purified is applied to layers of CsClsolution with final concentrations of 0.75, 1.5, 2.5 and 3.25 M CsCl. Atthe end of centrifugation the gradient is eluted into fractions.Fractions containing HBsAg may be identified by UV absorbance at 280 nmor by testing dilutions of the fractions with the AUSZYME kit. The HBsAgband is at a density of 1.17 to 1.23 g/cm³.

[0052] The solution containing the purified HBsAg is sterile filteredbefore being used to make a vaccine formulation.

[0053] Purification from the yeast cell lysate is complex as the antigenis produced intracellularly and a series of separation techniquesdesigned to eliminate different types of (yeast) contaminants arenecessary to obtain pure bulk antigen. The steps of purification areimportant, as the product to be purified is a lipoprotein particlecontaining multiple copies of the surface antigen polypeptide and thisstructure must be maintained throughout the purification process. It isa particularity of this process that it yields surface antigen particleswhich are fully immunogenic without the need for further chemicaltreatment to enhance immunogenicity (compare EPO 35435).

[0054] The details of the production process are further described inEuropean Patent 0199698.

EXAMPLE 2 Production and Characterization of Yeast-Derived HBsAg by aThiomersal Free Process

[0055] 1. Production and Purification of Yeast-Derived HBsAg

[0056] 1.1 Outline of the Production Process

[0057] Hepatitis B surface antigen may be produced by fermentation of anappropriate strain of Saccharomyces cerevisiae, for example thatdescribed in Harford et. al. (loc. cit.).

[0058] At the end of large-scale fermentation of the recombinant yeaststrain, the cells are harvested and broken open in the presence of amild surfactant such as Tween 20. The surface antigen is then isolatedby a multistep extraction and purification procedure exactly asdescribed above in Example 1 up to the step of the first gel permeationon Sepharose 4B.

[0059] 1.2 Thiomersal-Free Purification Process

[0060] In the thiomersal free process the following two changes havebeen introduced compared to the process described in Example 1.

[0061] 1. The elution buffer at the 4B gel permeation chromatographystep no longer contains thiomersal.

[0062] 2. Cysteine (2 mM final concentration) is added to the eluatepool from the anion exhange chromatography step.

[0063] It was found that omission of thiomersal from the 4B gelpermeation buffer may result in precipitation of the HBsAg particlesduring the CsCl density gradient centrifugation step with loss ofproduct and aggregation or clumping of the recovered antigen.

[0064] Addition of cysteine at 2 mM final concentration to the eluatepool from the preceding anion exchange chromatography step preventsprecipitation and loss of antigen during CsCl density centrifugation.

[0065] Cysteine is a preferred substance for this treatment as it is anaturally occurring amino acid and can be removed at the subsequentdesalting step on a gel permeation column using Sepharose 4BCLFF as thecolumn matrix.

[0066] There are no other changes in the manufacturing process comparedto the process described in Example 1.

[0067] The thiomersal free process yields bulk antigen of a purity andwith properties comparable to antigen from the process of Example 1.

[0068] 1.2a

[0069] The thiomersal added to the 4B buffer at 50 μg/ml is thought todecompose and the resulting ethyl mercury may attach covalently to freesulphydryl groups on cysteine residues of the protein. The proteincontains 14 cysteine residues of which 7 are located between positions101 and 150.

[0070] This region of the protein is believed to be located at thesurface of the particle and contain the major antigenic region of HBsAgincluding the immunodominant a region and the recognition site for theRF1 monoclonal antibody (Waters J et al, Postgrad. Med. J., 1987:63(Suppl. 2): 51-56.and Ashton-Rickardt and Murray J. Med. Virology, 1989:29: 196). Antigen purified with thiomersal present in the 4B gelpermeation buffer contains about 0.5-0.6 μg mercury at the end of thepurification process. This mercury is not fully removed by simpledialysis.

[0071] In one experiment, 0.56 μg Mercury per 20 μg protein was measuredon bulk antigen preparation. This preparation was dialysed for 16 hoursat room temperature against 150 mM NaCl, 10 mM NaPO₄ buffer pH 6.9. Atthe end of dialysis, a concentration of 0.33 μg Hg per 20 μg protein wasmeasured.

[0072] In contrast, dialysis in the presence of a reducing agent such asL-cysteine at 0.1 to 5.0 mg/ml, DTT at 50 mM or 2-mercaptoethanol at 0.5M, followed by a second dialysis to remove the reducing agent, resultsin reduction of the mercury content of the antigen preparation to lessthan 0.025 μg Mercury per 20 μg protein. This is the lowest limit ofdetection of the method.

[0073] The mercury content was determined by absorptionspectrophotometry. The antigen is diluted in a solution containing 0.01%w/v of potassium bichromate (K₂Cr₂O₇) and 5% v/v of nitric acid.Standard solutions are prepared with thiomersal as the mercury source.The atomic absorption of sample and standard solutions is measured aftervaporisation in a vapour generator, with a mercury-specific cathode at253.7 nm. Atomic absorption of the dilution liquid is measured as blank.The mercury content of the sample is calculated via the calibrationcurves obtained from the standard solutions. Results are expressed as pgof mercury per 20 μg of protein.

[0074] 1.3 Production of Thiomersal Free Bulk Antigen

[0075] The process steps for purification of bulk antigen are shown inFIG. 1.

[0076] 1.4 Composition of Vaccine Formulated Without Thiomersal.

[0077] A typical quantitative composition for a hepatitis B vaccinewithout preservative and formulated from antigen prepared by thethiomersal free process is provided in Table 1. TABLE 1 ConstituentAmount per ml Active constituent - Protein of which at least 95% 20 μgis HBsAg Aluminium hydroxide (adsorbent) 0.95 mg (expressed as Al₂O₃)Sodium chloride 9.0 mg (maximum) Disodium phosphate dihydrate 0.98 mgSodium dihydrogen phosphate dihydrate 0.71 mg Water for injection q.s.ad 1.0 ml

[0078] The composition may be varied by the addition of 3D-MPL and/orother adjuvants.

[0079] 2. Characterization of Bulk Antigen and Vaccine Produced by theThiomersal Free Process

[0080] 2.1. Tests and Assays on Purified Bulk Antigen

[0081] 2.1.1 Basis of Comparison

[0082] Three lots of bulk antigen were prepared by the thiomersal freeprocess according to this example (Example 1.2) and are identified asHEF001, HEF002 and HEF003. These were compared to a lot of bulk antigen(HEP2055) prepared by the previous process (as described in Example 1)in the presence of thiomersal.

[0083] 2.1.2 Tests and Assays on Bulk Antigen

[0084] The three bulk antigen lots produced by the thiomersal freeprocess were tested and the results are summarised in Table 2.

[0085] Protein content was measured by the method of Lowry et al (J.Biol. Chem. 1951:193:265).

[0086] Endotoxin content was measured by a Limulus gel clottingtechnique using a commercially available kit from Cape Cod Associates,704 Main St., Falmouth, Mass. 02540, USA. The reagent is standardizedagainst the US Pharm. Endotoxin Reference Standard.

[0087] Tween 20 was measured by the method of Huddleston and Allred (J.Amer. Oil Chemist Soc., 1965:42:983).

[0088] HBsAg content was measured by the commercially available AusZYMEkit from Abbott Laboratories, One Abbott Park Road, Abbott Park, Ill.60064, USA. Assay procedure B of the manufacturer was employed. A batchof bulk antigen purified by the process containing thiomersal was usedas a standard to establish the dose response curve.

[0089] Polysaccharides were measured by the method of Dubois et al(Anal. Chem. 1956:28:350).

[0090] Lipids were measured using a commercially available kit(Merkotest Total Lipids 3321) from E.Merck, B.P. 4119, Darmstad D-6100,Germanny.

[0091] DNA content was measured by the Threshold method using apparatusand reagents available from Molecular Devices Corp., Gutenbergstraβe 10,Ismaning, Munich, Germany.

[0092] The values found in the tests and assays are in the range seenfor bulk antigen lots manufactured using thiomersal in the elutionbuffer of the Sepharose 4B gel permeation step, with the exception ofthe antigenic activity by ELISA. The values for this measurement for thethree HEF preparations are higher (1.63-2.25) than that found for thebulk antigen lot HEP2055 which has a ELISA/protein ratio of 1.13. TheELISA/protein ratios measured by the AUSZYME kit for thiomersalcontaining batches of bulk antigen are generally about 1.0 and withinthe range 0.8-1.2 and very rarely exceed 1.4.

[0093] 2.1.3 SDS-PAGE Gel Analysis

[0094] The bulk antigen preparations were assayed by SDS-PAGE analysisin reducing conditions and Coomassie blue staining. All samples showed amajor band at 24K With traces of a dimer protein. The samples werejudged to be of high purity (>99% pure) as assessed by the absence ofvisible bands of contaminating proteins.

[0095] Samples (1 μg) of the bulk antigen preparations were assayed bySDS-PAGE in reducing and non-reducing conditions and silver staining(FIG. 2). In reducing conditions the samples showed an intense bandmigrating at 24K with traces of dimer and multimeric forms. The gelpatterns are indistinguishable from that of HEP2055 as comparator. Thesamples were also run in non-reducing conditions. In these conditionsless of the material migrates at 24K and the amount of polypeptidemigrating at dimeric and multimeric positions is increased. Thethiomersal free bulk antigen lots appear to have a somewhat higherdegree of polymerisation than the comparator HEP2055 lot.

[0096] The identity of the 24K polypeptide revealed by Coomassie blue orsilver staining was confirmed by Western blotting with rabbit polyclonalantibodies raised against plasma HBsAg. The bulk antigen preparationsshow a major band at 24K together with dimeric and trimeric forms. Thetechnique reveals minor traces of breakdown products of the surfaceantigen protein. There are no differences between the bulk antigenprepared by the thiomersal free process and the HEP2055 lot.

[0097] The presence of residual yeast proteins was assayed by SDS-PAGEanalysis in reducing conditions and Western blotting with rabbitpolyclonal antiserum raised against yeast proteins (FIG. 3). Thetechnique is qualitative and does not permit quantitation of theimpurities.

[0098] A constant band pattern is shown over the three bulk antigen lotsprepared by the thiomersal free process and the HEP2055 lot with oneexception.

[0099] A heavily staining band present at ±23K in the HEP2055 bulkantigen is virtually absent in the 3 HEF preparations. The Westernblotting shows that the thiomersal free purification process results ina purer antigen product. TABLE 2 Results of tests and assays onpurified, thiomersal free bulk antigen RESULT TEST HEF001 HEF002 HEF003HEP2055 PH 6.8 6.8 6.8 6.8 Protein content by Lowry 1312 μg/ml 888 μg/ml913 μg/ml 995 μg/ml Endotoxin content <0.25 EU <0.25 EU <0.25 EU <0.25EU Tween 20 content 7.1 μg 6.6 μg 7.4 μg 5.8 μg Antigenic activity byELISA 2957 μg/ml 1505 μg/ml 1486 μg/ml 1128 μg/ml ELISA/protein ratio2.25 1.69 1.63 1.13 Polysaccharide content 0.33 μg 0.35 μg 0.33 μg 0.34μg Lipid content 13.7 μg 12.8 μg 12.9 μg 11.8 μg DNA content byThreshold <1 pg <1 pg <1 pg <1 pg

[0100] 2.1.4 Other Biotechnical Tests and Assays

[0101] 2.1.4.1 DNA Content

[0102] The DNA content of the 3 bulk antigen lots was measured by theThreshold method (Molecular Devices Corp). The amounts measured wereless than 10 μg DNA per 20 μg protein (Table 2); the same level of DNAcontent seen with bulk antigen produced by the current approved process.

[0103] 2.1.4.2 Amino Acid Composition

[0104] The amino acid composition of the three HEF bulk antigen lots wasdetermined after acid hydrolysis with 6N HCl by chromatography of theamino acids on an ion exchange column with post column ninhydrindetection. Proline and tryptophan were not determined. The results aregiven in Table 3.

[0105] The compositions found are in good agreement with that determinedon HEP2055 and with the expected composition derived from the DNAsequence. Although the number of glycine residues measured for HEP2055is close to the expected composition, a value of 16 to 17 residues ismore usually measured for bulk antigen preparations. The mean number ofcysteine residues found is the expected 14, showing that no extracysteines are bound to the particle as a result of the treatment at theCsCl gradient step.

[0106] 2.1.4.3 Quantification of Free Cysteine

[0107] The quantity of free cysteine present in bulk antigenpreparations obtained according to the method described was measuredafter oxidation of the particles with performic acid without prior acidhydrolysis. Oxidised free cysteine residues were separated on an ionexchange column with post column detection by ninhydrin. The limit ofdetection of cysteine by this method is 1 μg per ml.

[0108] No free cysteine could be measured in the 3 HEF antigenpreparations when tested at the initial protein concentrations given inTable 2.

[0109] The technique measures both free cysteine residues present in thebuffer and cysteine residues which are attached to the HBsAg protein bydisulphide bonding but which do not form part of the polypeptidesequence.

[0110] 2.1.4.4 N-Terminal Sequence Analysis

[0111] The presence of possible protein contaminants and degradationproducts in the three bulk antigen lots produced by the modified processwas assessed by N-terminal sequence analysis based on Edman degradation.The N-terminal sequence MENITS . . . of the HBsAg protein was detectedwith no interference from other sequences. The N-terminal methionine wasalso confirmed to be 60-75% blocked by acetylation, as observedpreviously for HBsAg polypeptide produced by the routine process. TABLE3 Amino acid composition of HBsAg Amino Mean Expected acid HEF001 HEF002HEF003 comp. HEP2055 comp. Asp 11.3 11.3 11.3 11.3 11.5 10 Thr 17.5 17.417.2 17.4 17.8 17 Ser 21.4 21.6 21.4 21.5 20.9 23 Glu 11 11 11 11.0 10.59 Pro nd nd nd nd 24 Gly 17.1 16.8 16.7 16.9 14.6 14 Ala 7.5 7.4 7.4 7.47.2 6 Cys 12.3 14.95 14.9 14.1 13.2 14 Val 10.9 11 10.9 10.9 10.7 11 Met6.8 6.7 7.1 6.9 7.1 6 Ile 12.3 12.4 12.5 12.4 12.2 16 Leu 26.3 26.6 26.226.4 26.7 33 Tyr 6.8 6.8 6.8 6.8 7 6 Phe 13.8 13.9 13.8 13.8 13.9 15 His3 2.8 3.3 3.0 3.3 1 Lys 4 4 3.9 4.0 4.2 3 Arg 5.7 5.8 5.7 5.7 6.1 5 Trpnd nd nd nd 13

[0112] 2.1.4.5 Laser Light Scattering Analysis

[0113] Particle size comparisons were made by laser light scatteringbetween the HBsAg particles produced using the modified process and theHEP2055 reference lot (Table 4).

[0114] The mean molecular weights determined show good consistencybetween the preparations. TABLE 4 HBsAg particle molecular weights bylaser light scattering Antigen lot MW (Daltons) HEF001 3.07 × 10⁶ HEF0022.76 × 10⁶ HEF003 2.76 × 10⁶ HEP2055 3.34 × 10⁶

[0115] 2.1.4.6 Electron Microscopy

[0116] The bulk antigen preparations were examined by electronmicroscopy after fixation and staining with uranyl acetate.

[0117] The particles observed were similar in all the samples andconformed to the ±20 nm subspherical or cobblestone-like particlestypical of HBsAg. The particles observed in the 3 HEF lots wereindistinguishable from HEP2055.

[0118] 2.1.5 Immunological Analyses

[0119] 2.1.5.1 Reactivity with RF1 Monoclonal Antibody

[0120] The three bulk antigen preparations were tested for theirreactivity with the RF1 monoclonal antibody by ELISA inhibition assay.The RF1 monoclonal antibody has been shown to protect chimpanzeesagainst challenge with HBV and is considered to recognize a protectiveconformational epitope on the HBsAg particle (Iwarson S et al, 1985,J.Med, Virol., 16: 89-96).

[0121] The RF1 hybridoma may be propagated in the peritoneal cavity ofBalbC mice or in tissue culture.

[0122] Ascitic fluid diluted at {fraction (1/50000)} in saturationbuffer (PBS containing 1% BSA, 0.1% Tween 20) was mixed 1:1 with variousdilutions in PBS of the HBsAg samples to be tested (final concentrationsranging between 100 μg and 0.05 μg/ml).

[0123] Mixtures were incubated in Nunc Immunoplates (96U) for 1 hr at37° C. before being transferred for 1 hr at 37° C. onto plates coatedwith a standard preparation of HBsAg. The standard HBsAg preparation wasa lot of bulk antigen (Hep 286) purified by the thiomersal containingprocess. After a washing step with PBS containing 0.1% Tween 20,biotin-conjugated sheep anti-mouse IgG diluted {fraction (1/1000)} insaturation buffer was added to and incubated for 1 hr at 37° C. After awashing step, streptavidin-biotinylated peroxydase complex diluted{fraction (1/1000)} in saturation buffer was added to the same wells andincubated for 30 min at 37° C. Plates were washed and incubated with asolution of OPDA 0.04%, H₂O₂ 0.03% in 0.1M citrate buffer pH 4.5 for 20min at room temperature. The reaction was stopped with 2N H₂SO4 and theoptical densities (O.D.) were measured at 490/630 nm and plottedgraphically.

[0124] The IC50, defined as the concentration of antigen (inhibitorconcentration) that inhibits 50% of the antibody binding to coated HBsAgwas calculated using a 4 parameters equation and expressed in ng/ml.

[0125] A series of HEP antigen lots including HEP2055 were also tested,together with the Herpes simplex gD antigen as negative control. Theassay measures the ability of each test antigen to inhibit binding ofRF1 to a standard antigen preparation (HEP286) bound to microtitreplates.

[0126] Table 5 gives the concentrations of each antigen found to inhibit50% of RF1 binding to the fixed antigen. TABLE 5 Inhibition of bindingof RF1 monoclonal antibody to HBsAg Bulk antigen IC50 (ng/ml)* HEP2863834 HEP673 3437 HEP720 3150 HEP2055 2384 HEF001 468 HEF002 574 HEF003540

[0127] The results show that 4 to 7 fold less HEF antigen is required toinhibit RFI binding (Table 5). This shows that antigen prepared by themodified process has an increased presentation of the RF1 epitopecompared to HEP bulk antigen.

[0128] The same type of inhibition assay was performed using human serafrom Engerix B™ vaccinees instead of the RF1 mAb and did not revealdifferences between the HEP antigen lots and the HEF antigens.

[0129] 2.1.5.2. Affinity of Binding to Monoclonal RF1

[0130] The kinetic parameters of RF1 monoclonal antibody binding to the3 HEF antigen lots and to HEP2055 were measured by surface plasmonresonance using a Biacore 2000 apparatus from Amersham PharmaciaBiotech, Amersham Place, Little Chalfont, Bucks, UK.

[0131] The kinetic parameters measured were:

[0132] ka: the association rate constant (M⁻¹S⁻¹)

[0133] kd: the dissociation rate constant (S⁻¹)

[0134] Ka: the equilibrium or affinity constant (M⁻¹)

[0135] where ${Ka} = \frac{ka}{kd}$

[0136] The values found are given in Table 6. TABLE 6 Affinity constantsof RF1 binding to HBsAg ka kd Ka Bulk antigen (×10⁻³) (×10⁵) (×10⁻⁷)HEF001 6.81 3.21 21.97 HEF002 6.89 3.73 18.83 HEF003 7.39 4.67 15.80HEP2055 3.31 6.30 5.31

[0137] The three HEF antigen lots gave similar association/dissociationconstants and binding affinity values. In contrast HEP2055 has a weakeraffinity for binding to RF1.

[0138] This is consistent with the results from the ELISA inhibitionassay which showed that antigen prepared by the thiomersal free processhad an increased presentation of the RF1 epitope.

[0139] 2.2. Test and Assays on Vaccine Formulated with Antigen Producedby the Modified Process

[0140] The three HEF antigen lots were adsorbed onto aluminium hydroxideand formulated as vaccine according ot the composition as shown inTable 1. The presentation is the adult dose in vials (20

g antigen protein in 1 ml). The lots are identified as DENS001A4,DENS002A4 and DENS003A4.

[0141] Vaccine potency was measured by an in-vitro antigen content assayusing the Abbott Laboratories AUSZYME ELISA kit and a classical lot ofvaccine formulated with 50

g/ml thiomersal as standard. Vaccine potency was measured using method Bas described in PharmaEuropa Special Issue Bio97-2 (December 1997). Thethree HEF lots give high values for antigen content, nearly twice thestated content of 20 μg antigen protein.

[0142] 2..2..1 Reactivity of DENS Vaccine with RF1 Monoclonal Antibody

[0143] The antigenicity of the adsorbed vaccine was further tested in aninhibition assay with RF1 monoclonal antibody. The assay measures theability of the vaccine sample to inhibit RF1 binding to fixed bulkantigen (HEP286).

[0144] Ascitic fluid diluted at {fraction (1/50000)} in saturationbuffer (PBS containing 1% BSA, 0.1% Tween 20) was mixed 1:1 with variousdilutions in PBS of the vaccine samples to be tested (concentrationranging between 20 μg and 0.05 μg/ml).

[0145] Mixtures were incubated in Nunc Immunoplates (96U) for 2 hr at37° C. with agitation before being transferred onto HBsAg coated plates.The HBsAg preparation used for coating was a lot of bulk antigen (Hep286) purified by the thiomesal containing process. These plates are thenincubated for 2 hr at 37° C. with agitation. After a washing step withPBS containing 0.1% Tween 20, biotin-conjugated sheep anti-mouse IgGdiluted {fraction (1/1000)} in saturation buffer was added and incubatedfor 1 hr at 37° C. After a washing step, streptavidin-biotinylatedperoxydase complex diluted {fraction (1/1000)} in saturation buffer wasadded to the wells and incubated for 30 min at 37° C. Plates were washedand incubated for 20 min at room temperature with a solution containingOPDA 0.04%, H₂O₂ 0.03% in 0.1M citrate buffer pH 4.5. The reaction wasstopped with 2N H₂SO4 and optical densities (O.D.) were measured at490/630 nm and plotted graphically.

[0146] The IC50, defined as the concentration of antigen (inhibitorconcentration) that inhibits 50% of the antibody binding to coated HBsAgwas calculated using a 4 parameters equation and expressed in ng/ml.

[0147] Vaccine prepared from bulk antigen produced by the modifiedprocess was compared to Engerix B™ vaccine formulated from classical HEPbulk antigen and without thiomersal as preservative.

[0148] The assays were run in triplicate.

[0149] The results are given in Table 7 and show that about half thequantity of DENS vaccine is required to achieve 50% inhibition of RF1binding as compared to preservative free Engerix B™ vaccine. Thisreflects an increased presentation of the RF1 epitope on the HEF/DENSantigen and is consistent with the tests done with RF1 antibody on thepurified bulk antigen. TABLE 7 Inhibition of RF1 binding by formulatedvaccine IC-50 (ng/ml)⁽¹⁾ Experiment Vaccine lot 1 2 3 Mean DENS001A4 913662 603 726 DENS002A4 888 715 521 708 DENS003A4 817 685 582 695ENG5100A2 1606 1514 1481 1534 ENG3199B9 1329 1170 1286 1262 ENG3328A91417 1194 1334 1315

[0150] 2..2..2 Immunogenicity of DENS Vaccine in Mice

[0151] A study was performed in Balb/C mice in order to compare theimmunogenicity of the three DENS consistency lots to Engerix B™ producedaccording to the current antigen manufacturing process and formulatedwith thiomersal.

[0152] The following lots were tested:

[0153] # DENS001A4

[0154] # DENS002A4

[0155] # DENS003A4

[0156] # ENG2953A4/Q as reference

[0157] Briefly, groups of 12 mice were immunised intramuscularly twiceat 2 weeks interval with vaccine doses corresponding to {fraction(1/10)} (2 μg) or {fraction (1/50)} (0.4 μg) of the adult human dose.Antibody response to HBsAg and the isotypic profile induced byvaccination were monitored from sera taken at day 28.

EXPERIMENTAL DESIGN

[0158] Groups of 12 Balb/C mice were immunised intramuscularly in bothlegs (2×50 μl) on days 0 and 15 with the following vaccine doses: TABLE8 Groups and vaccine dose Antigen Group Vaccine Volume dose 1 DENS001A4100 μl   2 μg 2 → Diluted 5× in PO4/NaCl 100 μl 0.4 μg 3 DENS002A4 100μl   2 μg 4 → Diluted 5× in PO4/NaCl 100 μl 0.4 μg 5 DENS003A4 100 μl  2 μg 6 → Diluted 5× in PO4/NaCl 100 μl 0.4 μg 7 ENG2953A4/Q 100 μl   2μg 8 → Diluted 5× in PO4/NaCl 100 μl 0.4 μg

[0159] On days 15 (2 weeks post I) and 28 (2 weeks post II) blood wastaken from the retroorbital sinus.

[0160] For the design of this experiment (4 formulations×2 doses with 12mice per group), the power was estimated a priori with the PASSstatistical program. The PASS (Power and Sample Size) statisticalprogramme was obtained from NCSS, 329 North 1000 East, Kaysville, Utah84037. For the 2 way analysis of variance, a 2.5 fold difference of GMTbetween formulations with an alpha error of 5% should be detected with apower >90%.

[0161] Results

[0162] Serology:

[0163] Humoral responses (Total Ig and isotypes) were measured by ELISAassay using HBsAg (Hep286) as coating antigen and biotin conjugatedanti-mouse antibodies to reveal anti-HBs antibody binding. Only post 11sera were analysed.

[0164] Table 9 shows the mean and GMT anti-HBs Ig antibody responsesmeasured on individual sera at 2 weeks post II

[0165] Comparable antibody responses are induced by the DENS andclassical hepatitis B formulations: GMT ranging between 2304 and 3976EU/ml for the DENS lots compared to 2882 EU/ml for SB Biologicalshepatitis B monovalent vaccine (Engerix B™) at the 2 μg dose, and GMTranging between 696 and 1182 EU/ml for the DENS lots compared to 627EU/ml for SB Biologicals hepatitis B monovalent vaccine (Engerix B™) atthe 0.4 μg dose.

[0166] As expected a clear antigen dose range effect is observed for allformulations at the 2 μg and 0.4 μg doses with a 3 to 6 fold differencein GMTs.

[0167] Four non responder mice (titers<50EU/ml) were observed withoutclear links to the antigen doses or lots used for the injection (Groups1, 2, 3 and 8; one mouse per group). Based on statistical analysis(Grubbs Test) these mice were discarded from further analysis. TABLE 9Antibody response in mice at day 28 (2 weeks post II ELISA TITERS (lg)Group Vaccine Dose Number Mean GMT 1 DENS001A4   2 μg 11 3466 2971 2 0.4μg 11 1283 1182 3 DENS002A4   2 μg 11 2436 2304 4 0.4 μg 12 984 786 5DENS003A4   2 μg 12 4583 3976 6 0.4 μg 12 997 696 7 ENG2953A4/Q   2 μg12 3999 2882 0.4 μg 11 737 627

[0168] Statistical Analysis:

[0169] A 2 way-analysis of variance was performed on the anti-HBs titersafter log transformation of post II data, using the vaccines (4 lots)and antigen doses (2 μg and 0.4 μg) as factors. This analysis confirmedthat a statistically significant difference was observed between the twoantigen doses (p value<0.001) and did not show any significantdifference between the vaccine lots (p value=0,2674). As previouslymentioned the power was estimated a priori and the design of theexperiment was such that a 2.5 fold difference of GMT with a alpha errorof 5% could be detected between formulations with a power>90%.

[0170] Isotypic profile:

[0171] Table 10 shows the isotypic repartition (IgG1, IgG2a and IgG2b)calculated from an analysis on pooled sera at post II.

[0172] As expected, a clear TH2 response is induced by these alum basedvaccines as mainly IgG1 antibodies are observed.

[0173] No difference is observed between the DENS lots or SB Biologicalshepatitis B monovalent vaccine in term of isotypic profile. TABLE 10Repartition of IgG isotypes in pooled day 28 sera Isotype (%) GroupVaccine Dose IgG1 IgG2a IgG2b 1 DENS001A4   2 μg 91 4 5 2 0.4 μg 87 8 53 DENS002A4   2 μg 97 2 1 4 0.4 μg 87 6 7 5 DENS003A4   2 μg 98 1 1 60.4 μg 93 4 3 7 ENG2953A4/Q   2 μg 88 8 4 0.4 μg 88 9 3

EXAMPLE 3 Formulation of Combined Vaccines

[0174] The bulk antigen of the invention is particularly suitable forformulation in a combined vaccine comprising IPV.

[0175] Stability studies performed on initial lots of a combinedDTPa-HBV-IPV vaccine indicated a decline in potency of the IPVcomponent, particularly of type 1 poliomyelitis antigen, when using anin vitro immunoassay (determination of D-antigen content by ELISA) andan in vivo rat potency test. No potency loss was observed for type 3.For type 2, the potency loss was within the expected range (not morethan 10% loss per year of storage).

[0176] Studies were initiated to determine the cause of this loss ofpotency in the combined DTPa-HBV-IPV vaccine. From the observation thatthe stability of IPV in SB Biologicals' DTPa-IPV vaccine is satisfactory(not more than 10% antigen content loss per year of storage), it wasconcluded that the HBV component was likely to be responsible for theinstability of IPV in the DTPa-HBV-IPV vaccine.

[0177] The HBV component used in the initial DTPa-HBV-IPV formulation isthe purified r-DNA, yeast-derived HBsAg also used for the manufacture ofSB Biologicals hepatitis B monovalent vaccine and prepared as describedin Example 1.

[0178] As a first attempt to determine which element in the HBVcomponent was deleterious to IPV, the HBsAg bulk was analysed for thepresence of thiomersal. It has been previously found (Davisson et al.,1956, J. Lab. Clin. Med 47: 8-19) that thiomersal used as preservativein DTP vaccines “was detrimental to the poliomyelitis virus” in aDTP-IPV combination. This observation was considered by vaccinemanufacturers who have replaced thiomersal with other preservatives toformulate their IPV-containing vaccines. More recently, the effect ofthiomersal on IPV potency under conditions of long-term storage at +4°C. was reinvestigated. The loss of potency of type 1 polio virus antigento undetectable levels after 4-6 months was reported (Sawyer, L. A. etal. 1994, Vaccine 12: 851-856).

[0179] Using atomic adsorption spectroscopy, approximately 0.5 μg ofmercury (Hg) per 20 μg of HBsAg was detected in antigen purifiedaccording to Example 1.

[0180] This amount of mercury (as thiomersal and ethylmercury chloride,the thiomersal degradation product) can reduce to undetectable levelsthe ELISA response for D-antigen type 1 content in an IPV bulkconcentrate incubated at 37° C. for 7 days.

[0181] A method was established to release mercury present in the HBsAgbulk. It was postulated that mercury could be bound to thiol groups onthe HBsAg particle and could therefore be released in the presence ofreducing agents. After experimentation with other reducing agents,L-Cysteine was selected as the agent for release of mercury from theHBsAg particle. After dialysis of HBsAg bulk against saline solutioncontaining 5.7 mM L-Cysteine, no mercury was detected in the retentate(detection limit of the testing method: 25 ng Hg/20 μg HBSAg). Thedialysed antigen was mixed with IPV bulk concentrate and the stabilityof the type 1 virus was assessed by measuring the D-antigen contentafter incubation at 37° C. for 7 days. The IPV bulk concentratenon-mixed and mixed with HBsAg not treated with cysteine were used ascontrols. The reference ELISA titre was obtained on the samples storedat +2° C. to +8° C. for 7 days.

[0182] The results are summarised in Table 11: TABLE 11 D-antigencontent (type 1)⁽¹⁾ Sample 7 days/4° C. 7 days/37° C. Loss IPV(non-mixed) 31.6 24.2 23% IPV + HBsAg not treated 31.1 18.1 42% IPV +HBsAg-cysteine-treated 31.4 27.6 12% IPV + thiomersal (1 μg/ml) 30.511.0 74%

[0183] The data obtained on these laboratory preparations clearlydemonstrate that the stability of the type 1 polio virus issignificantly improved if HBsAg is treated with cysteine to removeresidual mercury prior to mixing with IPV.

[0184] The data presented above also show a loss of D-antigen content of23% for the reference IPV preparation after incubation for 7 days at 37°C. This confirms the inherent instability of the type 1 Mahoney poliovirus, as previously reported (Sawyer, L. A. et al. (1994), Vaccine 12:851-856).

[0185] Although commercial lots of DTPa-HBV-IPV and DTPa-HBV-IPV/Hibvaccines have been prepared using a dialysis process with 5.7 mML-Cysteine to remove residual mercury and preserve the stability of IPV,the dialysis process is not suited to large scale production andinvolves a series of supplementary steps to prepare thiomersal ormercury free HBsAg. In contrast, the HBsAg of the present invention,prepared without thiomersal, may be directly used in formulations ofcombined vaccines especially those containing IPV.

[0186] 4. Summary

[0187] The previously used process for purification of yeast-derivedsurface antigen contains a gel permeation step where the mercurycontaining anti-microbial compound thiomersal is included in the elutionbuffer to control bioburden.

[0188] The thiomersal is not completely cleared during the subsequentsteps of the process so that about 1.2 μg thiomersal per 20 μg proteinis present in the purified bulk antigen.

[0189] In order to produce a completely thiomersal (mercury) free bulkantigen the purification process has been altered at two steps.

[0190] Thiomersal is omitted from the elution buffer at the 4B gelpermeation step.

[0191] Cysteine (2 mM final concentration) is added to the eluate poolfrom the anion exchange chromatography step. This prevents precipitationof antigen during CsCl density gradient centrifugation.

[0192] There are no other changes to the production process.

[0193] The bulk antigen produced by the modified process has beencharacterized. Physico-chemical tests and assays show that thethiomersal free antigen is indistinguishable in its properties fromantigen produced by the previously used process. The antigen particleshave the same constituents.

[0194] The identity and integrity of the HBsAg polypeptide is unaffectedby the modified process as judged by SDS-PAGE analysis, Western blottingusing polyclonal anti-HBsAg antibodies, N-terminal sequence analysis andamino acid composition. Electron microscopy and laser light scatteringanalysis show that the particles are of the typical form and sizeexpected for yeast-derived HBsAg. Analysis by Western blotting withanti-yeast protein serum shows that the antigen produced by thethiomersal free process has a similar pattern of contaminating yeastproteins. However, the amount of a contaminating band migrating at 23Kis greatly reduced in the 3 HBsAg lots produced using the modifiedprocess.

[0195] Immunological analyses show that the thiomersal free particleshave an increased antigenicity. The particles are more reactive with theAbbott AUSZYME kit (containing a mixture of monoclonal antibodies)giving ELISA/protein ratios of 1.6 to 2.25. This increased antigenicityis also shown with the protective RF1 monoclonal antibody. About 4 to 7fold less thiomersal free antigen is required to inhibit RF1 binding toa standard fixed antigen. The thiomersal free and classical antigeninhibition of binding curves fall into two distinct families. Thisdifference is also shown by measurements of the binding affinityconstant for RF1 using surface plasmon resonance. The binding affinitiesof the thiomersal free preparations are 3 to 4 fold higher compared tothe lot of classical bulk antigen.

[0196] The bulk antigen preparations were formulated as vaccine byadsorption onto aluminium hydroxide and without preservative.

[0197] Testing for in vitro potency using the Abbott AUSZYME ELISA kitand thiomersal containing SB Biologicals hepatitis B monovalent vaccineas standard showed that high in vitro potency values were obtained. Theantigen content measured by this test was nearly double the stated valueof 20 μg protein per ml.

[0198] An increased reactivity of vaccine prepared from thiomersal freeantigen was also seen in an inhibition assay with RF1 monoclonalantibody for binding to fixed antigen. About half the quantity ofthiomersal free vaccine was required to give 50% inhibition of RF1binding to fixed antigen as compared to antigen purified by thepreviously used process and formulated without preservative.

[0199] This increased antigenicity of the thiomersal free vaccine withrespect to RF1 is consistent with the results from the in vitro potencytest (antigen content) and with the RF1 antibody tests performed on thebulk antigen preparations.

[0200] A mouse immunogenicity test was performed using priming andbooster vaccinations two weeks apart and doses of 2 and 0.4 μg antigen.Mice were bled on day 28, 14 days after the booster. The sera wereanalysed for antibody titre and isotype composition. A clear antigendose effect was observed for the two doses administered but there was nostatistically significant difference in the response in terms ofantibody titres (GMT) between thiomersal free and preservative freevaccines.

[0201] No substantial differences were observed in the isotype profiles.

1 A hepatitis B vaccine comprising a purified hepatitis B antigen, theantigen comprising less than 0.025 μg mercuy per 20 μg protein, theantigen being obtainable by purification in the presence of a reducingagent having a free —SH group. 2 A vaccine according to claim 1 which iswithout preservative. 3 A vaccine according to claim 2 wherein thepreservative is thiomersal. 4 A vaccine according to any of claims 1-3wherein the hepatitis antigen is adsorbed onto aluminium hydroxide. 5 Avaccine according to any preceding claim wherein the hepatitis B antigenis obtainable by subjecting a crude hepatitis B preparation to thefollowing steps: (a) gel permeation chromatography; (b) ion-exchangechromatography; and (c) mixed with a reducing agent having a free —SHgroup 6 A vaccine according to any preceding claim wherein the reducingagent is cysteine, glutathione,dithiothreitol or β-mercaptoethanol. 7 Avaccine according to claim 6, wherein the reducing agent is cysteine. 8A vaccine according to claim 7 wherein the cysteine is added to a finalcentration of between 1-10 mM. 9 A vaccine according to claim 8 whereinthe cysteine is added to a final centration of about 2 mM. 10 A vaccineaccording to any preceding claim wherein the antigen is purified in thepresence of thiomersal before treatment with the reducing agent. 11 Avaccine according to any of claims 1-9 wherein the purified antigen iscompletely free of thiomersal. 12 A vaccine according to claim 11 whichis a thiomersal free vaccine. 13 A vaccine composition according to anypreceding claim in conjunction with an adjuvant. 14 A vaccinecomposition according to claim 13 wherein the adjuvant is an aluminiumsalt. 15 A vaccine composition according to claim 14 wherein thealuminium salt is aluminium hydroxide 16 A vaccine composition asclaimed in claim 13-15 which comprises a TH-1 inducing adjuvant. 17 Avaccine composition according to claim 16 wherein the TH1-inducingadjuvant is selected from the group comprising: 3-DMPL, QS21, 3-DMPL andQS21, and aCpG oligonucleotide. 18 A vaccine composition as claimed inany preceding claim which additionally comprises one or more of theantigens selected from the group consisting of: diptheria toxoid (D),tetanus toxoid (T) acellular pertussis antigens (Pa), inactivated poliovirus (IPV), haemophilus influenzae antigen (Hib), hepatitis A antigen,herpes simplex virus (HSV), chlamydia, GSB, HPV, streptococcuspneumoniae and neisseria antigens. 19 Use of a hepatitis antigencomprising less than 0.025 μg mercury per 20 μg protein in thepreparation of a preservative free hepatitis vaccine, the antigen beingobtainable by purification in the presence of a reducing agent having afree —SH group. 20 A method for producing a hepatitis B antigen suitablefor use in a vaccine, the method comprising purification of the antigenin the presence of a reducing agent having a free —SH group, wherein theantigen is purified in the presence of thiomersal before treatment withthe reducing agent. 21 A method for producing a hepatitis B antigenaccording to claim 20, wherein the purified antigen product comprisesless than 0.025 μg mercury per 20 μg protein 22 A method of producing ahepatitis B antigen according to claim 20 or 21 wherein a crudehepatitis B antigen preparation is: (a) subjected to gel permeationchromatography; (b) subjected to ion-exchange chromatography; and (c)mixed with a reducing agent having a free —SH group 23 A method ofproducing a hepatitis B antigen according to claim 20-22 wherein thereducing agent is cysteine, glutathione, dithiothreitol orβ-mercaptoethanol. 24 An immnunogenic hepatitis B antigen, the antigencomprising less than 0.025 μg mercury per 20 μg protein, the antigenbeing obtainable by purification in the presence of a reducing agenthaving a free —SH group and wherein the antigen is purified in thepresence of thiomersal before treatment with the reducing agent. 25 Avaccine comprising a hepatitis B antigen as claimed in claim 24 26 Amethod, antigen or vaccine composition according to any preceding claimwherein the hepatitis B antigen is a surface antigen. 27 A method oftreatment and/or prophylaxis of hepatitis B virus infections, the methodcomprising administering to a human or animal subject suffering from orsusceptible to hepatitis B infection a safe and effective amount of avaccine according to any of claims 1-18 and 25.